Functionalization and in vitro study of cytotoxic salan titanium(IV)-bis-chelates

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Functionalization and in vitro study of cytotoxic salan titanium(IV)-bis-chelates

Dissertation

zur Erlangung des akademischen Grades

des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz

vorgelegt von Tiankun Zhao

Tag der mündlichen Prüfung: 13. Nov. 2015

Vorsitzender: Prof. Dr. A. Marx

Prof. Dr. A. Bürkle 1. Referent:

2. Referent:

Dr. T. Huhn

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I would like to express my deepest gratitude to my supervisor: Dr. Thomas Huhn for providing me the chance and guidance to pursue my chemistry career. Dr. Huhn spend a lot of effort and time throughout this research. His strict scientific attitude, passion at chemistry and easygoing manner influence and benefit me greatly in my further career.

I would like to thank Prof. Tanja Gaich, Prof. Ulrich Groth, Ms. Malin Bein, Ms. Angelika Früh and Ms. Milena Quentin for their assistance during my study.

A big thanks to Dr. Dmytro Sysoiev for your time and sharp hawk eyes on this dissertation. Special thanks is also extended to Dr. Mikhail Kabdulov, Dr. Peter Schmitt for your support with X-ray and discussions; Fabian Schneider, Mareike Rapp, Lea Spitzer, Nikolas Schön, Tim Strohmeier for doing your bachelor theses with me; Nils Rotthowe, Johannes Bayer for doing your master practice with me; Tetiana Druzhenko for doing your exchange study with me; all other members from AG Gaich and previous AG Groth. It is a pleasure to work with you!

Special thanks to:

Konstanz Research School Chemical Biology (KoRS-CB) for providing me the scholarship and research funding.

Prof. Andreas Marx and Prof. Alexander Bürkle for helpful discussions about this dissertation.

Dr. Heike Brandstädter and Katharina Magerkurth for your assistance.

Mr. Ulrich Haunz and Mrs. Anke Friemel for helping with the NMR measurements.

My family and friends for your solid support during my studies.

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Abstract

Cancer known as malignant neoplasm is one of the diseases that cause highest disease incidence and death rate in the world ^[1-2]. The discovery of the anticancer effects of cisplatin and its clinical introduction in the 1970s represents an important step in the history of the development of metal-based cancer drugs.

For decades, seeking for novel metal complexes with improved pharmacological properties became one of the major tasks for chemists. The discovery of titanocene dichloride and Budotitan demonstrated that titanium(IV) complexes are potential novel anti-tumor agents. However, the main drawback is their fast hydrolysis in the presence of water, which results in the stagnation of their clinical trials. Salan type Ti(IV) complexes receive much attention due to their broad anti-tumor spectra and enhanced stability towards hydrolysis. After we introduced pyridine-2, 6-dicarboxylic acid (dipic) as a second chelator, the aqueous stability of the resulting heptacoordinate complexes was greatly enhanced without the loss of cytotoxicity.^[3-4]

In this dissertation, the focus is on expanding and further modifications of the concept of salan Ti(IV)- bis-chelates and the study of their biological and hydrolysis behavior. The main content of the thesis targets the development of functionalized novel salan ligands, their respective Ti(IV) complexes and the direct functionalization of Ti(IV) complexes by transition metal catalyzed C-C and C-N bond forming reactions. By incorporation of different functional groups, our research is addressing from the following aspects: synthesis and characterization of Ti(IV) complexes; complex functionalization; study of complexes’ cytotoxicity, stability and other possible applications. The detailed work of this dissertation is structured in four major parts:

Part 1: Starting from Salicylaldehyde, we synthesized four sulfonamide functionalized salan ligands and their salan Ti(IV)-bis-chelates. Structure characterization by HRMS, ¹H, ¹³C NMR spectroscopy and X- ray diffraction revealed the sulfonamide group to be not bound to the titanium. Bio evaluation of their anticancer activity against the human carcinoma HeLa S3 cell line and human hepatocellular carcinoma Hep G2 cell line reveals that most of the synthesized complexes exhibit moderate to potent activity.

Among them, complexes 7b and 7c showed excellent activity against the two tested cell lines. Their IC50

values range from 0.5 to 0.9 μM against Hela S3 and 1.0 to 1.8 μM against Hep G2 cell lines, respectively, which is 5 to 7 times higher than that of cisplatin (IC50 range from 2.0 to 4.8 μM).

Part 2: Staring from diketene, we synthesized two “folate like” precursors which contain alkynyl group, 6-((4-ethynylphenyl-amino)-methyl)-pteridine-2,4-diamine (27) and (2,4-diaminopteridin-6-yl)methyl

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4-ethynylbenzoate (28). Attempts to conjugate these two compounds to azido salan Ti(IV)-bis-chelate via a Cu-catalyzed azide-alkyne-cycloaddition (CuAAC) failed due to poor solubility. 2-amino-6- (bromo-methyl)-pteridin-4(3-H)-one (39) was prepared in an effort to gain access to a functionalized folic acid derivative. It turned out that the derivatives electrophilicity was too limited for further modifications via nucleophilic substitution towards titanium complexes.

Part 3: Through copper mediated CuAAC reaction, we synthesized four estrogen containing salan Ti(IV)-bis-chelates with different poly-ethylene glycol (PEG) linkers (mono-, di-, tri- and tetra-ethylene glycol), all these four complexes are characterized by IR, HRMS, ¹H and ¹³C NMR spectroscopy.

Hydrolysis studies monitored by ¹H NMR spectroscopy revealed that complex 59a was stable in the presence of 1000 equiv. D2O for at least 24 h. Preliminary bioevaluation showed that estrogen mono ethyleneglycol linked salan Ti(IV)-bis-chelate lost its cytotoxicity against estrogen receptor (ER) negative Hela S3 and Hep G2 cell lines. Further bioevaluation against ER positive cell lines (MCF-7 and HT-29) is currently under investigation.

Part 4: Functionalization of both ligand systems independently allowed application of palladium based coupling reactions on the already assembled titanium complexes. The bioevaluation for anti-cancer activity against HeLa S3 and Hep G2 cell lines revealed that most of the complexes exhibited moderate to potent activity. Complexes 65a, 65f, 66i, 66j showed excellent bioactivity with their IC50 values range from 0.5-1.6 μM against Hela S3 and 0.7-3.0 μM against Hep G2 cell lines, which is 2 to 5 times higher than that of cisplatin. Hydrolysis studies demonstrated that complexes with alkynyl group on the dipic were stable in the presence of 1000 equiv. D2O for weeks, whereas complexes having the alkynyl functionality on the salan were comparable less stable than our previously reported 2,4-dimethy salan Ti(IV)-bis-chelate.^[5]

Keywords: Cancer; Titanium complex; Sulfonamide; Folic acid; CuAAC Reaction; Estrogen, Targeted drug delivery; Sonogashira reaction; Alkyne

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Zusammenfassung

Krebs, eine maligne Neoplasie, ist eine der Krankheiten mit dem höchsten Verbreitungsgrad und Sterberate weltweit.^[1-2] Die Entdeckung der antineoplastischen Wirkung von Cisplatin und Einführung der klinischen Anwendung in den 1970er-Jahren stellt einen wichtigen Schritt in der Geschichte der Entwicklung Metall-basierter Krebsmedikamente dar. Seit Jahrzehnten entwickelt sich die Suche nach neuen Metallkomplexen mit verbesserten pharmakologischen Eigenschaften zu einer umfassenden Aufgabe für Chemiker. Die Entdeckungen von Titanocendichlorid und Budotitan haben gezeigt, dass sich Titan(IV)-Komplexe potentiell als neue Antitumormittel eignen. Ein großer Nachteil dieser Komplexe ist ihre schnelle Hydrolyse in Gegenwart von Wasser, was die Stagnation der klinischen Phase-II-Studien zur Folge hatte. An Salan-Ti(IV)-Komplexen herrscht erhebliches Interesse aufgrund ihres breiten Antitumorspektrums und ihrer erhöhten Hydrolysestabilität. Durch Einführung von Pyridin- 2,6-dicarbonsäure (dipic) als zweitem Chelatligand wurde die Hydrolysestabilität der so erhaltenen, siebenfach koordinierten, Komplexen massiv verstärkt ohne Verlust der Cytotoxizität.^[3-4]

Der Fokus dieser Dissertation liegt auf dem Ausbau und weiterer Modifikation des Konzepts der Salan Ti(IV)-dipic-bis-chelatkomplexe und des Studiums ihres biologischen und Hydrolyseverhaltens. Ein Großteil dieser Arbeit zielt daher auf die Entwicklung von neuartigen funktionalisierten Salanliganden und ihren jeweiligen Ti(IV)-Komplexen, sowie auf die direkte Funktionalisierung von Komplexen durch übergangsmetallkatalysierte CC- und CN-Bindungsknüpfungen ab. Durch den Einbau von verschiedenen funktionellen Gruppen wurden folgende Aspekte untersucht: Synthese und Charakterisierung von Ti(IV)-Komplexen, Funktionalisierung von Komplexen, Zytotoxizität der Komplexe, Stabilität und mögliche weitere Anwendungen. Diese Dissertation ist in vier Hauptteile gegliedert:

Teil 1: Ausgehend von Salicylaldehyd wurden vier Sulfonamid funktionalisierte Salanliganden und deren salanTi(IV)-bis-chelatkomplexe synthetisiert. Die Strukturen wurden durch HRMS, ¹H,¹³C NMR- Spektroskopie und Röntgendiffraktometrie charakterisiert. Es konnte gezeigt werden dass die Sulfonamidgruppe nicht an das Titan gebunden ist. Die Evaluation ihrer Cytotoxizität gegen die menschlichen Zervixcarcinomzelllinien HeLa S3 und Hepatocarzinomzelllinen Hep G2 zeigte, dass die meisten der synthetisierten Komplexe moderate bis starke Bioaktivität aufwiesen. Die Komplexe 7b und 7c zeigten eine ausgezeichnete Aktivität gegenüber den beiden getesteten Zelllinien. Die IC50-Werte liegen im Bereich von 0,5 bis 0,9 μM für Hela S3- und 1,0 bis 1,8 μM für Hep G2-Zelllinien, was um 5 bis 7 mal höheren IC50-Werten als jenen von Cisplatin (IC50 Bereich von 2,0 bis 4,8 uM) entspricht.

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Teil 2: Ausgehend von Diketen wurden zwei alkinfunktionalisierte folatartige Vorläuferverbindungen synthetisiert, 6-((4-Ethinylphenyl-amino)-methyl)-pteridin-2,4-diamin(27) und (2,4-Diaminopteridin-6- yl)methyl 4-ethinylbenzoat 28. Versuche die Verbindungen durch Cu(I) katalysierte Azid-Alkin- Cycloaddition (CuAAC) an azidfunktionalisierteTi(IV)-Komplexe zu ligieren scheiterten aufgrund geringer Löslichkeit der Reaktanden. Es wurde 2-Amino-6-(bromomethyl)-pteridin-4(3-H)-on 39 synthetisiert um Zugang zu funktionalisierbaren Folsäurederivaten zu erhalten, die Verbindung erwies sich jedoch als zu wenig elektrophil um weiter durch nucleophile Substitution modifiziert werden zu können und Zugang zu den entsprechenden Titanverbindungen zu erhalten.

Teil 3: Durch Kupfer(I) katalysierte CuAAC-Reaktion wurden vier Östrogen verknüpfte SalanTi(IV)- Komplexe unter Verwendung verschiedener PEG-Linker (Mono-, Di-, Tri-, Tetraethylenglycol) dargestellt. Die Verbindungen wurden durch IR, HRMS, ¹H and¹³C NMR Spektroskopie charakterisiert.

Hydrolysestudien unter Verwendung von ¹H NMR Spektroskopie zeigten dass alle vier Komplexe in Gegenwart von 1000 equiv. D2O für mindestens 24 h stabil sind. Die vorläufige Evaluation der Bioaktivität der Verbindungen zeigte, dass die Komplexe keine Zytotoxizität gegenüber Östrogen- Rezeptor-negativen Hela S3 und Hep G2 Zelllinien aufweisen.

Teil 4: Die unabhängige Funktionalisierung beider Ligandsysteme ermöglichte die Anwendung Palladium katalysierter Kupplungsreaktionen an den fertigen Komplexen. Die Evaluation der Cytotoxizität gegenüber HeLa S3 und Hep G2 Zellen zeigte mittlere bis starke Aktivität der meisten Komplexe. Die Komplexe 65a, 65f, 66i, 66j zeigten hervorragende Aktivität mit IC50-Werten im Bereich von 0,5-1,6 μM gegen Hela S3 und 0,7-3,0 μM gegen Hep G2 Zelllinien, die damit 2 bis 5-mal höher liegt als die Cytotoxizität von Cisplatin. Hydrolysestudien zeigten dass die Komplexe mit alkinfunktionalisierter Dipicolinsäure in Gegenwart von 1000 equiv. D2O über Wochen stabil bleiben, während Komplexe mit Alkinfunktionalität des Salanliganden vergleichsweise weniger stabil sind als die bereits zuvor bekannten 2,4-Dimethylsalan-Ti(IV)-bis-Chelate.^[5]

Schlüsselworte: Krebs; Titan-Komplexe; Sulfonamide; Folsäure; Clickreaktion; Östrogen, Targeted drug delivery; SonogashiraReaktion, Alkin

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With the change of human living style and deterioration of environment, cancer has become one of the serious diseases that cause serious harm to human health.^[1] According to estimation from the World Health Organization, there are over 70 million people in the world are suffering from the cancer till 2014.

The growing rate is about 8.7 million people annually. Among them, 3.3 million in developed countries and 5.4 million in developing countries. American Cancer Society (ACS) predicted that due to the global population growth and aging population, cancer infection rate will reach to 19.3 million per annual in 2025. Lung, liver, stomach and colon cancer are major diseases that cause deaths. Therefore, the development of new anticancer drugs have always been the focus for medicinal chemists.^[6]

Chemotherapy is currently the main method for cancer treatment and is widely used in clinical treatments. Chemotherapeutic agents such as cisplatin, alkylating agents, anti-tumor antibiotics and natural products are commonly used. Development of these medications prolongs the patient’s life and improves their life quality. However, an anti-tumor effect can be achieved only when the drug accumulation in tumor cells reaches to or above a certain level,however, due to the low selectivity of these chemotherapeutic drugs used in the clinic, it is difficult to limit the cytostatic effect so that only cancer cells are destroyed. Low selectivity and the unspecific effect towards healthy cells and tissue often cause unpleasant side effects. Furthermore, drug resistance arising from longtime clinical use urgently calls for the development of new anti-tumor agents with enhanced cytotoxicity and spectrum, but less side effects. The development of non-platinum anticancer drugs, especially titanium complexes as antitumor drugs draws more and more attention and has become one of the hottest area in medicinal chemistry.^[7]

In 1969, Rosenberg first reported the discovery of the anticancer effects of Cisplatin, which is an important step in the history of the development of cancer drugs. Cisplatin was introduced into clinical use in the 1970s. Since then, it received huge success. Since then, cisplatin is still one of the most important chemotherapeutic agent used against different solid tumors, including testicular, bladder, ovarian, head and neck cancer. The mechanism is based on the formation of platinum-DNA adducts, which leads to distortion of the helical DNA structure, thus inhibiting the DNA replication.^[8-9] More platinum based drugs have been developed and went into clinical use. E.g. Carboplatin, Oxaplatin, Nedaplatin and Lobaplatin.^[10] (Figure 1)

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Figure 1. Platinum based anti-tumor drugs.^[8-10]

Platinum based drugs are usually associated with severe side effects because of poor specificity.

Cisplatin has systemic toxicities like nephrotoxicity, neurotoxicity, ototoxicity and emetogenesis inflicting serious disorders or injuries on the patients during the treatment, which badly restrict its efficacy. More and more efforts have been spent on developing new anti-tumor drugs. In recent years, a large number of compounds containing platinum as well as non-platinum metals were reported to be effective against tumors both in vivo and vitro.^[11] These compounds comprise main group metals such as Gallium, Germanium, Tin and Bismuth, early transition metals like Titanium, Vanadium, Niobium, Molybdenum and Rhenium as well as late-transition metals like Ruthenium, Rhodium, Iridium, Platinum, Copper and Gold.

Titanium and Titanium chemistry

Titanium is the ninth most abundant element in the earth’s crust and is the second most abundant transition metal (after iron).Titanium is the first member of the 3d transition series and has an electronic configuration of 3d24s2. The most stable oxidation state is IV, lower oxidation state compounds in a range of -I, 0, II, III, are also known.^[12] The most commonly use of titanium is in the form of its dioxide-TiO2. TiO2 is widely used as a white pigment in the manufacture of paint and paper and as filler in rubber and plastics, and has found wide spread application in skin care and cosmetics products and as sun blocker in sunscreens.^[13]

The common coordination number of titanium is six, although four-, five-, seven-, and eight- coordinate compounds are known.^[14] However, due to the high ionic charge to radius ratio, Ti(IV) compounds are difficult to prepare from aqueous solutions. Most reported Ti(IV)reagents are not water compatible and the syntheses are performed in organic solvent with more or less rigorous exclusion of water.^[15]

In 1979, Köpf and Köpf-Maier opened a new chapter in medicinal chemistry with the revolutionary discovery of the first metallocene-based anticancer agent, titanocene dichloride, Cp2TiCl2. Since then,

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titanocene dichloride derivatives and β-diketonato complexes such as budotitane have been studied as anti-tumor compounds; both compounds went into clinical trials as anticancer drugs.^[16-17](Figure 2)

Figure 2. Anti-tumor titanium complex: Titanocene dichloride and Budotitane.^[16-17]

In 2008, Meléndez's group reported the use of amino acids (L-cysteine, L-methionine, L-penicillamine) as ligands to synthesize titanocene derivatives. Unfortunately, the obtained complexes are biologically inactive against colon cancer HT-29 cell line.^[18] While titanocene dichloride itself has an IC50 of 450 µM against HT-29 cell line. (Figure 3)

Figure 3. Titanocenes complexes with amino acids as ligands. The amino acids are bound to the titanium via their carboxylates.^[18]

In 2004, Tacke’s group reported the synthesis of six indolyl-substituted titanocenes through reaction of various 6-indolylfulvenes with super hydride (LiBEt3H), followed by transmetalation with titanium tetrachloride (TiCl4).

Figure 4.Indolyl functionalized titanocenes.^[19]

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Incorporation of titanocenes with biological active indolyl motif leads to more effective cytotoxic behavior against CAKI-1 cell line than the well-studied Titanocene Y (bis[(p- methoxybenzyl)cyclopentadienyl]titanium(IV) dichloride).^[19](Figure 4)

In 2010, Meléndez's group reported steroid-functionalized titanocenes as potential anticancer drugs.

The IC50 values are as low as 10 µM against HT-29 (colon cancer) and MCF-7 (breast cancer cells) which is more cytotoxic and selective than titanocene dichloride. This study provides a good example for using sex steroids as pendant groups for the anti-cancer drug, potentially resulting in target specific anti-cancer drugs for hormone dependent cancers.^[20](Figure 5)

Figure 5. Steroid functionalized titanocene derivatives.^[20]

Several other laboratories also have published new titanium complexes for evaluation as potential anticancer drugs.^[21-23] Since their cytotoxicity is measured in different assays or against different cell lines, it is difficult to compare their efficacy. In general, most of the Cp2TiCl2 derivatives reported are benchmarked against and are more active than Cp2TiCl2.^[24-25]

The salen type ligands are widely used in coordination chemistry. This class of compounds are named after the simplest example, N,N'-bis(salicylidene)ethylenediamine, which are referred to as “Salen”.

Reduction of the “Salen” affords a new tetradentate ligand, generally known as “Salan”. Salan ligands are conformationally flexible and adopt a variety of geometries. Functionalization of the ligands with different substituents could enhance the biological properties. Many “Salen” and “Salan” metal complexes (such as Cu, Zn, Mn, Fe, Al, Zr, Ni, Ru, Cr, Lanthanide metals) have been reported to show activity as anti-cancer agents,^[26] antibacterial agents,^[27-28] antiviral agents,^[29] fungicide agents^[30] and other biological properties.^[31-32]

The first Salan Ti(IV) complex was reported in 2001 by Walsh and co-workers.^[33] In 2007, Tshuva and co-workers reported that in HT-29 and ovarian OVCAR-1 cells, their cytotoxicity was higher than reference compounds Cp2TiCl2, (bzac)2Ti(OⁱPr)2 and Cisplatin. In their work, they presented that salan Ti(IV) complexes exhibits a transferrin independent celluar uptake process.^[34] (Table 1)

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Table 1. Cytotoxicity comparsion of different classes of titanium complexes in dependence of added transferrin.^[34]

N N

Ti OiPr O O

R₂

R₂ R₁ R₁ R₃

R₃

[L1Ti(OⁱPr)₂]: R₁= R₃=t-Bu, R₂= H [L2Ti(OⁱPr)₂]: R₁= R₃= Me, R₂= H [L3Ti(OⁱPr)₂]: R₁= H, R₂= R₃= Me N

N

Ti OiPr O O

R R R

R

OiPr

R= H,t-Bu Walsh and co-workers 2001

OiPr

Tshuva and co-workers, 2007

Reagent^a IC50 [μM]

HT-29 OVCAR-1 HT-29 + Tr^b OVCAR-1+Tr^b Cp2TiCl2 710 ± 120 780 ± 90 460 ± 40 520 ± 30 (bzac)2Ti(OⁱPr)2 53± 1 53 ± 1 57 ± 1 65 ± 1

L1Ti(OⁱPr)2 nontoxic nontoxic nontoxic nontoxic

L2Ti(OⁱPr)2 12±1 12±1 20±3 40±4

L3Ti(OⁱPr)2 12±1 14±1 16±3 15±3

Cisplatin 33 ± 3 17 ± 4

a 72 h incubation. ^b Tr = Transferrin.

In 2009, our group reported the synthesis of a series of halogen (F, Br, Cl ) substituted salan ligands and their metalation with titanium tetra-isopropoxide (Ti(OⁱPr)4) to give racemic C2 symmetrical Ti(IV) complexes. Their bioevaluation and apoptosis study reaveals that a chloro substituted complex is the first titanium complex that combines high cytotoxicity against HeLa S3 and Hep G2 cells and the selective induction of apoptotic cell death.^[35] (Table 2)

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Table 2. Halogen substituted salan Ti(IV)-isopropoxides.^[35]

R^a Hela S3 IC50 [μM] Hep G2

F 1.6 ± 0.1 2.2 ± 0.2

Cl 5.3 ± 0.2 4.0 ± 0.2

Br 13 ± 1 40 ± 6

Cisplatin 1.2 ± 0.4 3.0 ± 1.3

a 48 h incubation.

In 2012, our group reported the use of 2, 6-pyridinedicarboxylic acid (dipic) for functionalization of salan Ti(IV)-bis-chelates via ligand exchange.^[5] The resulting heptacoodinate complex is redox inactive, stable on silica gel and has improved aqueous stability compared to its isopropoxide precursor.

Table 3. Functionalization of salan Ti(IV)-isopropoxide by dipic to afford hepta-coordinated salan Ti(IV)-bis-chelate.^[4-5]

Complex IC50 [μM](Hela S3)^a IC50[μM] (Hep G2)^a t1/2b

Ti(IV)-isopropoxide 2.3 ± 0.1 2.1 ± 0.1 10 h

Ti(IV)-bis-chelate 4.4 ± 0.4 3.4 ± 0.3 ≫ 2 weeks

Cisplatin 1.2 ± 0.4 3.0 ± 1.3

a 48 h of incubation. ^bTime of 50% decomposition of Ti(IV)-isopropoxide and Ti(IV)-bis-chelate in presence of 1000 equiv. of D2O.

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Besides this, the Ti(IV) complex is highly toxic against Hela S3 and Hep G2 cell lines and with an enhanced anti-tumor efficacy in a mouse cervical-cancer model.^[5] The hydrolytic study was carried out by time-resolved ¹H NMR in the presence of 1000 equiv. D2O at 37 ^oC. We found that this type of salan Ti(IV)-bis-chelate is more stable than its alkoxide precursor. We incubated the Ti(IV)-bis-chelate at 37

oC for over two weeks, no precipitates or degradation was detected. (Table 3)

Based on this study, Zhuravlev and co-worker reported in 2015 a solid-phase based ⁴⁵Ti radiolabeling methodology for the synthesis of salan ⁴⁵Ti(IV)-bis-chelate as ⁴⁵Ti-PET imaging probe.^[36] The ex vivo evaluation was carried out on HT-29 colorectal tumor bearing mice. This salan ⁴⁵Ti(IV)-bis-chelate was found to metabolize in the liver then to the gall bladder and then to the intestines. This complex showed a fast hepatobiliary excretion pattern with almost no renal clearance. (Figure 6). This study provided an example of using promising titanium compounds to become a useful tool for accelerating clinical translation. Furthermore, Ti/⁴⁵Ti-based anticancer agents with fast and significant tumor uptake would combine chemotherapeutic effect with diagnostic and molecular imaging features, ushering the Ti-based metallodrugs into the growing field of theranostics.

Figure 6. (Left) Solid-phase based synthesis of salan ⁴⁵Ti(IV)-bis-chelate. (Right) Representative PET/CT images following iv injection of 1.6 MBq salan ⁴⁵Ti(IV)-bis-chelate at various time points after injection. The arrows point at the ROI (region of interest) corresponding to red, heart/blood; orange, liver; blue, gall bladder; pink, cecum; green, colon/feces.^[36]

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2. Description of proposed research

Based on the concept of stabilization of titanium(IV) salan complexes by bis-hetero-chelation, previously developed in our group, we will investigate the scope and limitations of further functionalization of heptacoordinate Ti(IV) species. We hope to access enhanced cytotoxicity and develop versatile methodology for direct combinatorial modification to this type of complex, aiming for molecule libraries construction and screening for potential anti-cancer agents. Potential application in other fields such as materials science will also be our research target. (Scheme 1)

Scheme 1. Graphical representation of intended research.

Our work will be carried out into the following four directions:

(1) Synthesis of sulfonamide substituted salan ligands and construction of corresponding salan Ti(IV) - bis-chelates.

(2) Introduction of alkynyl and azido functional groups to salan Ti(IV)-bis-chelates and their incorporation with “Folic acid” and “Estrogen” via copper catalyzed CuAAC reaction.

(3) Introduction of iodine to the salan Ti(IV)-bis-chelates. Reaction optimization of Pd catalyzed Sonogashira reaction for direct functionalization of the salan Ti(IV)-bis-chelate and its tolerance with different alkynes as well as further applications.

(4) Structure characterization; Bioevaluation; Stability study.

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3. Results and discussions

3.1. Synthesis and bioevaluation of sulfonamide salan Ti(IV)-bis-chelates 3.1.1 Sulfonamide and their anti-cancer properties

Sulfonamides are recognized as an important class of compounds that exhibit a broad spectrum of biological activities,^[37-38] particularly, a large number of these compounds have recently been reported to show substantial antitumor activities, and some of them have been investigated in clinical trials.^[39]Although these structurally novel compounds have a common chemical motif of aromatic /heterocyclic sulfonamides, there are a variety of mechanisms of their antitumor action, such as the disruption of microtubule assembly,^[40] cell cycle arrest in progression,^[41] inhibition of carbonic anhydrase,^[42] of methionine amino peptidase,^[43] of histone deacetylase,^[44]and of vascular endothelial growth factor.^[45]Table 4 shows a summary of recently developed sulfonamide antitumor drugs.

Table 4. Recently developed important sulfonamide anticancer agents.^[46-51]

Drug Therapeutic use Structure

Batabulin^[46] Anticancer

Pazopanib^[47] Anticancer

Tamsulosin^[48] Anticancer

ABT-751^[49] Anticancer

E-7070^[50] Anticancer

E-7820^[51] Anticancer

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E7010, the breakthrough discovery, reported by Yoshino et al. in 1992, is a tubulin polymerization inhibitor that reversibly binds to the colchicine site of r-tubulin, and showed consistent growth- inhibitory activities against a panel of 26 human tumor cell lines (IC50 = 0.06 - 0.8 μg/mL).^[52] E7010 inhibited the tumor growth in mice by 60-99 % following oral administration at doses of 25-100 mg/kg daily for 8 days. It is also effective against vincristine, cisplatin, and 5-fluorouracil resistant P388 cell lines in mice.^[53] (Figure 7)

Figure 7. Anticancer drug candidate E7010.^[52]

The sulfonamide group (R-SO2NH2) is the most important and widely used zinc binding agent. The majority of clinically used carbonic anhydrase inhibitors are sulfonamide derivatives, the mechanism studies illustrate the coordination of the deprotonated sulfonamide to the catalytic Zn⁺ ion, then substitution of water. This study highlighted that sulfonamide group is an ideal ligand of the CA active site.^[54] Since the first report in 1940, many aromatic sulfonamides have been synthesized and investigated for their CA inhibitory action.^[55] Benzene sulfonamides become the most and best- characterized targeting group.^[56-57]The benzene sulfonamide moiety is involved in the coordination of the Zn⁺ catalytic ion and the phenyl ring establishes several Van-der-Waals interactions with Gln92, Phe131, Leu198, Thr200 of human carbonic anhydrase II (HCA II) ^[58] (Figure 8)

S O O

-NH

N NH 2+Zn

N N NH NH HN

O O O^- H O

Glu106 Thr119

Phe131

Gln92

Val121 Leu198

Thr200

His96

His119

His94

Figure 8.Interaction of sulfonamide with Gln92, Phe131, Leu198, Thr200 of carbonic anhydrase II.

[58]

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Owa and co-workers reported another class of antitumor sulfonamides that block cell cycle progression of P388 murine leukemia cells in the G1 phase, a series of N-(7-indolyl)-benzene- sulfonamides having been obtained and evaluated as antitumor agents. The antitumor activities of these derivatives are enhanced by substitution with chloro or cyano substituents at the C-3 position of the indole moiety. Of the compounds examined, E7070 (indisulam) demonstrates significant antitumor activity both in vitro and in vivo against different human tumors.^[59](Figure 9)

Figure 9. Indolyl functionalized anti-tumor sulfonamides.^[59]

3.1.2 Sulfonamide incorporated organometallic complexes

In 2007, Supuran et al. reported the synthesis and bioevaluation of two CA recognizing benzene sulfonamides pharmacophores (ArSO2NH2) with attached ferrocenyl or ruthenocenyl moieties synthesized via CuAAC reaction. These metallocene derivatives exhibit nanomolar or low micromolar inhibitory activity against HCAI, and 9.7-80 nM and 10.3-85 nM against HCA II and HCA IX, respectively. Among them, the ruthenocenyl derivatives gives superior CA inhibition compared to the ferrocenyl compounds.^[60] (HCA = Human carbonic anhydrase) (Figure 10)

Figure 10. Synthesis of ArSO2NH2 derivatives with ferrocenyl or ruthenocenyl moieties via a CuAAC reaction.^[60]

In 2011, Ranninger’s group from the Autonomous University of Madrid reported the first synthesis of trans-N-sulfonamide substituted platinum complexes as anti-tumor agents, by incorporation of a

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non-coordinating sulfonamide group to the trans-platinum complexes. Two of the synthesized compounds exhibit comparable or enhanced bioactivity compared to cisplatin.^[61] (Figure 11)

Figure 11. First example of trans-N-sulfonamide (non-coordinating) platinum complexes.^[61]

Hou from Nanjing University reported the synthesis of half-sandwich cobalt and rhodium complexes with combination of carborane and non-coordinating N-sulfonamide. Both complexes inhibit the growth of the human NSCLC cell lines A549 and H460. Rh complex demonstrated to be more effective than Co complex. The growth inhibitory effect of the two complexes could be associated to alterations in the cell cycle profile of A549 cells without a significant induction of cellular apoptosis.^[62] Furthermore, the Rh complex altered the mRNA levels of CCND1, CCNE1 and PCNA, which are known to control G0/G1 phase of the cell cycle. However, the question whether the cytotoxicity effect is a result of the sulfonamide or carborane ligand were not explored in this study. (Figure 12)

Figure 12. Half sandwich cobalt and rhodium complexes with sulfonamide motif.^[62]

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Balanced modulation of multiple targets is an attractive therapeutic strategy in cancer treatment.

Combinations of drugs, either of targeted drugs or of cytotoxic plus targeted drugs, are frequently used in clinic therapy. Sulfonamide motifs are privileged functional groups in drug discovery and widely exist in the structures of many drugs as a key pharmacophore. This small molecule draws our attention to combine it with cytotoxic Ti(IV) complex. By introducing a small molecule antagonist, which has a variety of anti-cancer mechanism; we look forward to improve anticancer activity and tumor cell selectivity of Ti(IV) complexes.

Work of this chapter are carried out from the following directions:

(a) Design and synthesis of sulfonamide containing salan ligands.

(b) Synthesis of sulfonamide containing salan Ti(IV)-bis-chelates.

(c) Structure characterization, stability study and bioevaluation.

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3.1.3 Experiments and results

3.1.3.1 Synthesis of sulfonamide functionalized salan Ti(IV)-bis-chelates

Huntress et al. reported chlorosulfonation of arenes to give sulfonyl chlorides, and subsequent reaction with an amine affords aryl sulfonamides.^[63]We applied this method to the synthesis of sulfonamide salan ligands. As illustrated in Figure 13, starting from Schiff base 1 obtained from condensation of salicylaldehyde with aniline, chlorosulfonation of 1 gives sulfonyl chloride substituted salicylaldehyde 2 in 85 % yield.

Figure 13. Synthesis of sulfonyl chloride substituted salicylaldehyde 2.

The corresponding salan ligand was prepared according to the reported procedure.^[64] 2 reacted with four different secondary amines 3a-b-c-d (pyrrolidine 3a, piperidine 3b, azepane 3c and morpholine 3d), giving the corresponding sulfonamide substituted salicylaldehydes 4a-4d. Those were reacted with ethylenediamine, the resulting imines were isolated by filtration, and were directly reduced with sodium borohydride to afford the corresponding secondary amines, subsequent methylation by reductive methylation in the presence of HCHO, CH3COOH and NaBH4 gave the desired salans 5a-5d. Non-sulfonamide substituted salan ligand 5e was also prepared accordingly.

The results are summarized in Table 5, all ligands were characterized by ¹H NMR, ¹³C NMR, IR and mass spectroscopy.

Table 5. Synthesis of the sulfonamide salan ligands 5a-5d and salan ligand 5e.

Entry Amine Product Yield^a (%)

1 3a 5a 71

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2 3b 5b 32

3 3c 5c 38

4 3d 5d 28

5^b - 5e 67

a Isolated yield over 3 steps. ^b Salicylaldehyde 4e and ethylenediamine were used for preparation of 5e.

The metalation of the salan with Ti(OⁱPr)4 gives racemic Ti(IV) salan-isopropoxides 6a-6e in nearly quantitative yield as judged from ¹H NMR spectra recorded from the crude product. In contrast to Ti(IV)-alkoxides with alkyl or halogen substituted salans which are dark orange to red colored, complexes 6a-6d are virtually colorless. Only in the case of complex 6d, the isopropoxide was isolated and characterized. All other complexes were directly converted to the dipic derivatives 7a- 7e. The ligand exchange reaction was initiated by the addition of THF to a mixture of complexes and 1.2-2 equiv. dipic to afford salan Ti(IV) bis chelates 7a-7e in moderate yields. It is noteworthy that addition of less than 2 equiv. of dipic resulted in non-complete conversion for the synthesis of 7a-7d.

(Table 6)

Table 6. Synthesis of the sulfonamide containing salan Ti(IV)-bis-chelate 7a-7e.

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Entry^a ligand R Product Time (h) Yield^b(%)

1 _5a _7a ₁₂ ₃₃

2 _5b _7b ₁₅ ₄₀

3 _5c _7c ₁₂ ₄₀

4 _5d _7d ₁₆ ₄₅

5^c _5e H 7e 12 44

aAll reactions were performed under N2 on a 0.5 mmol scale, using Ti(OⁱPr)4 (1.1 equiv), dipic (2 equiv.) in THF (20 ml) at r.t. ^bIsolated yield over 2 steps. ^c dipic (1.1 equiv.) was used.

3.1.3.2 Demonstration of constitution

All obtained complexes were characterized by ¹H NMR, ¹³C NMR, IR, UV-Vis and MS spectroscopy. In the ¹H NMR spectra all complexes showed the characteristic signals for pseudo C2- symmetrical complexes, a coincidence of the signals of both halves of the salan backbone. In complex 7a, for example, The CH2 from the salan backbone give rise to a pair of AB signals in the region of 5.39 ppm and 3.37 ppm with coupling constant J = 16.0 Hz. NCH2 from the salan also results in a pair of AB type signals in the region of 3.32 ppm and 2.33 ppm, with coupling constants of each 8.0 Hz. In the ¹³C NMR spectrum, all carbon signals are observed.

Suitable single crystals of 7d and 7e were grown by slow diffusion of hexane into a saturated solution of 7d in acetone or of hexane into a saturated solution of 7e in dichloromethane. Both crystallize in the monoclinic space group C2/c. In the asymmetric unit one molecule 7d is accompanied by two molecules of acetone while 7e crystalizes without additional solvent. In 7e Ti(1), N(2) and C(12) are oriented on the two-fold axis which intersects the ethylenediamine bridge of the salan backbone between C(8) and C(8ⁱ). Consequently, the asymmetric unit contains only half a molecule. (Figure 14 for 7d. Figure 15 for 7e)

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Figure 14. X-ray crystal structure of heptacoordinate complex 7d accompanied by two molecules of acetone in the cell. Thermal ellipsoids are drawn at the 50% probability level. Hydrogen atoms are omitted for clarity.

Figure 15. X-ray crystal structure of heptacoordinate complex 7e. Thermal ellipsoids are drawn at the 50% probability level. Hydrogen atoms are omitted for clarity.

Selected bond length and angles are summarized in Table 7, crystallographic data are found in experimental part. With respect to the arrangement around the titanium centre 7d and 7e compare extremely well with the other known solid state structures of Ti(IV) salan dipicolinates.^[12-13] Both feature the pentagonal bipyramidal core structure of a heptacoordinate titanium centre with the phenolates in the apical position and the nitrogen atoms of the salan and the pyridine together with the carboxylates defining the equatorial plane. The bridging nitrogen atoms of the salan have the greatest deviation from the equatorial plane with 0.194(17) Å for N2 in 7d and 0.284(8) Å for N1 in 7e. The phenolate-Ti distance in 7e (1.8672(11) Å) is comparable to that in 7d (1.8586(15) Å and

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1.8648(15) Å). In both complexes this distance is slightly elongated compared to the 1.84 Å found for the 2,4-dimethylsubstituted salan in ref. 5 indicating that the p-sulfonamido substituted 7d as well as the unsubstituted homologue have a diminished donor strength compare with the 2,4- dimethylsubstituted complex. (Table 7)

Table 7. Selected bond lengths and angles for complexes 7d and 7e.

Complex 7d

Distances [Å] Angles [°]

Ti–O(11) 1.8586(15) O(11)–Ti–O(12) 170.21(7) O(11)–Ti–N(2) 80.98(6) Ti–O(12) 1.8648(15) O(11)–Ti–O(4) 95.06(6) O(12)–Ti–N(2) 92.01(6) Ti–O(4) 2.0361(15) O(12)–Ti–O(4) 89.38(6) O(4)–Ti–N(2) 73.19(6) Ti–O(2) 2.0551(14) O(11)–Ti–O(2) 89.21(6) O(2)–Ti–N(2) 145.08(6) Ti–N(1) 2.1941(18) O(12)–Ti–O(2) 92.75(6) N(1)–Ti–N(2) 143.21(6) Ti–N(2) 2.3457(17) O(4)–Ti–O(2) 141.42(6) O(11)–Ti–N(3) 91.22(6) Ti–N(3) 2.3757(18) O(11)–Ti–N(1) 94.33(7) O(12)–Ti–N(3) 80.22(6) O(12)–Ti–N(1) 95.36(7) O(4)–Ti–N(3) 144.99(6)

O(4)–Ti–N(1) 70.91(6) O(2)–Ti–N(3) 72.91(6) O(2)–Ti–N(1) 70.53(6) N(1)–Ti–N(3) 142.91(6)

N(2)–Ti–N(3) 73.88(6) Complex 7e

Distances [Å] Angles [°]

N(1)-Ti(1) 2.3695(14) O(1)#1-Ti(1)-O(1) 171.17(7) O(1)-Ti(1)-N(1) 80.46(5) N(2)-Ti(1) 2.178(2) O(1)#1-Ti(1)-O(2) 89.00(5) O(2)-Ti(1)-N(1) 73.05(5) O(1)-Ti(1) 1.8672(11) O(1)-Ti(1)-O(2) 93.84(5) O(2)#1-Ti(1)-N(1) 143.95(5) O(2)-Ti(1) 2.0345(12) O(2)-Ti(1)-O(2)#1 142.47(7) N(2)-Ti(1)-N(1) 143.46(3) O(1)-Ti(1)-N(2) 94.41(4) O(1)-Ti(1)-N(1)#1 92.41(5) O(2)-Ti(1)-N(2) 71.23(3) N(1)-Ti(1)-N(1)#1 73.08(7) O(1)#1-Ti(1)-N(1) 92.41(5)

The MeN–Ti distance in both complexes is unaffected by the substitution in the vicinity of the salan and is with 2.3457(17) and 2.3757(18) for 7d and 2.3695(14) Å for 7e in the range of 2.350(2) Å and 2.384(2) of the previous characterized complex.^[12] Dipicolinic acid acts as bis-anionic tridentate coordinating to the titanium-salan fragment via two of the carboxylate oxygen atoms and its pyridine N atom. In 7d both carboxylate groups of the dipic are pulled towards the Ti(IV) centre resulting in a twist out of plane with respect to the pyridine ring by 3.9(2)°. The Ti–carboxylate

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distances in 7d differ with 2.0361(15) Å and 2.0551(14) Å quite strongly whereas the Ti-O distances in the previous characterized complex is more balanced (2.043(1) and 2.046(1) Å).10 In 7e the Ti–

carboxylate distance is with 2.0345(12) Å considerably shorter.

3.1.3.3 Bioevaluation

All compounds were tested for their cytotoxicity in the human cervix carcinoma cell line HeLa S3 and the human hepatocarcinoma cell line Hep G2 in a AlamarBlue-based cytotoxicity assay.^[65] IC50

values are given as mean values from three independent experiments each done in four replicates.

The IC50 values of the synthesized compounds are summarized in Table 8. It was observed that compounds 7a, 7b and 7c exhibited good to excellent bioactivity and reached maximum inhibition, that is a cell viability of 0 % (For IC50 charts, see Figure 16). The ligands 5a-5e and dipic 8 were investigated for their cytotoxicity as well to answer the question if the measured cytotoxicity might be an effect of liberated ligand. Dose-response curves for the ligands 5a-5e were recorded in a concentration range comparable to that of the complexes. (For IC50 charts see Figure 16)

Two of the most active complexes 7b and 7c, with IC50 values below or close to 1 μmolar, are even more cytotoxic than cisplatin. Even though, the morpholinosulfonyl derivative 7d showed the anticipated enhanced aqueous solubility, its bioactivity against Hela S3 is greatly reduced when compared with 7a-7c and even completely vanished against Hep G2 cells. (Table 8)

Table 8. IC50-values obtained for 7a-7e by an AlamarBlue assay in Hela S3 and Hep G2 cells after 48h of incubation.

Complex IC50a [μM]

Hela S3 Hep G2

7a 3.1 ± 0.2 9.3 ± 0.2

7b 0.9 ± 0.1 1.8 ± 0.2

7c 0.5 ± 0.1 1 ± 0.1

7d 19 ± 4 nontoxic

7e 2.8 ± 0.5 3.7 ± 1.3

Cisplatin 2.0 ± 0.3 4.8 ± 1.2

a Values determined after 48h of incubation.

The ligands 5a-5e and dipic 8 were investigated for their cytotoxicity as well to answer the question if the measured cytotoxicity might be an effect of liberated ligand. Dose-response curves for the

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ligands 5a-5e were recorded in a concentration range comparable to that of the complexes. (For IC50

charts see Figure 16)

Figure 16. Comparison of viability of Hela S3 (left) and Hep G2 (right) cell after treatment with different concentrations of complexes 7a (red), 7b (black), 7c (yellow), 7d (green) and cisplatin (blue) (up) and ligands 5a (red), 5b (black), 5c (yellow), 5d (green) (down) after 48h of incubation.

One obvious difference observed is the lower solubility of the ligands 5a-5d compared to the corresponding complexes. While those were soluble close to the millimolar range during bio assays, the ligands showed limited solubility with the exception of 5d. As a result, the ligands dose-response plots did not follow a sigmoidal trend (A comparison of complex and ligand toxicity is given in Figure 17).

Instead, the curves for the ligands show a shallow slope and monotonously approach towards some cytotoxicity. At the 100 µmolar regime bioactivity was detected albeit the maximum cell viability never reached below 30%. Raising the concentration even further leads to severe solubility problems.

Only at this concentration regime where precipitation already was manifest highest cell viability was reached. Precipitation of 5a-5c was detected during the assay when concentrations were higher than 100 µmolar by microscopic control of the cell assay. This precipitation might account for the found cytotoxicity of the ligands at highly elevated concentrations.

Log c (M)

-10 -9 -8 -7 -6 -5 -4 -3 -2

% viability in Hela S3

0 20 40 60 80 100 120

5a 5b 5c 5c

Log c (M)

-10 -9 -8 -7 -6 -5 -4 -3 -2

% viability in Hep G2

0 20 40 60 80 100 120

5a 5b 5c 5d log c [M]

-10 -8 -6 -4

% viability in Hela S3

0 20 40 60 80 100

7a 7b 7c 7d cisplatin

log c [M]

-10 -8 -6 -4

% viability in Hep G2

0 20 40 60 80 100 120

7a 7b 7c 7d cisplatin

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Figure 17. Comparison of cell viability of Ti(IV) complexes 7a-7e with ligand 5a-5e against Hela S3 cells (left column) and Hep G2 cells (right column).

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80 100

7a 5a dipic

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80

7a 5a dipic

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80 100

7b 5b

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80 100

7b 5b

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80 100

7c 5c

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80 100

7c 5c

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80 100

7d 5d

log c [M]

-10 -9 -8 -7 -6 -5 -4 -3

% cell viability

0 20 40 60 80 100

7d 5d

log c [M] Log c/ [M]

-10 -9 -8 -7 -6 -5 -4 -3 -2

% Cell viability

0 20 40 60 80 100

7e 5e

Log c/ [M]

-10 -9 -8 -7 -6 -5 -4 -3 -2

% Cell viability

0 20 40 60 80 100

7e 5e

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In contrast, complexes 7a-7c showed potent cytotoxicity already at concentration in the µmolar regime with an inhibition rate of greater than 90%. The ligands 5a-5d and dipic, however, showed a less pronounced cytotoxicity in this concentration regime. In contrast to the behavior of the ligands, complexes 7a-7c lead to a complete loss of viability already at two orders of magnitude lower concentrations.

3.1.3.4 Stability study

We further carried out the stability test for 7a-7d by time-resolved ¹H NMR spectroscopy under the following conditions: Ti complex (15 µmol), 4-nitrotoluene (0.3 mg, 2.25 µmol, 0.15 equiv), D2O (0.27 ml, 1000 equiv.), [D8]-THF (0.4 ml). ¹H NMR was measured at 37 ^oC and incubated at 37 ^oC.

Data were gathered by monitoring the decrease in isolated signals of the titanium-bound salan backbone and increase in the free salan ligand backbone. (Figure 18) Integrals are normalized against the internal standard (4-nitrotoluene). Control measurements were done in the absence of 4- nitrotoluene and showed no significant difference in the hydrolysis rate and the product formed.

7a 7b

7d 7c

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Figure 18. Hydrolytic stability study of complexes 7a-7d (clockwise from upper left) at 37 ^oC investigated by time resolved ¹H NMR spectroscopy in aqueous (1000 equiv. D2O), [D8]-THF (0.4 ml) and 0.15 equiv. of 4-nitrotoluene as an internal reference. Spectra are shown from bottom to top measured at given time intervals after the addition of D2O. Signals marked A, B, C, D, E and F are Har of free salan ligand, Har of the complex, CarCH2 of the complex, D2O, [D8]-THF and CH3 of 4- nitrotoluene respectively.

The aim of the hydrolytic degradation study is to benchmark relative stability under controlled conditions, which are different from the hydrolysis in a biological environment. As shown in Table 9, all complexes exhibit moderate hydrolytic stability with their t1/2 range between 2.0-3.5 h, and thus are more prone towards hydrolysis than is our previously reported Ti(IV)-bis-chelate with t1/2 ≫ 2 weeks.^[5]The electron withdrawal nature of the sulfonamide renders the titanium more electrophilic and results in faster hydrolysis, probably due to less steric shielding because of a lack of ortho- substitution.

Table 9. Half-life data collected by time resolved ¹H NMR spectra of complexes 7a-7d.

Compound t½ (h)^a Compound t½ (h)^a

7a 2.0 7c 2.2

7b 3.5 7d 3.0

a Time for 50% hydrolysis, calculated based on pseudo first order kinetics.

Figure 19. (Left) Plots of hydrolysis vs. reaction time for 7a (■), 7b (♦), 7c (●), 7d (▲). (Right) Plots showing the liberation of free ligands vs. reaction time for 5a (■), 5b (♦), 5c (×), 5d (▲) during the hydrolytic stability study.

0 10 20 30 40 50 60 70 80 90 100

0 120 240 360 480 600 720 840 960

% Hydrolysis

t (min)

0 10 20 30 40 50 60 70 80 90 100

0 200 400 600 800 1000

% increase of ligand

t (min)

Functionalization and in vitro study of cytotoxic salan titanium(IV)-bis-chelates